Genomic sequencing is required for identification of tuberculosis transmission in Hawaii.

BACKGROUND: Tuberculosis (TB) caused an estimated 1.4 million deaths and 10.4 million new cases globally in 2015. TB rates in the United States continue to steadily decline, yet rates in the State of Hawaii are perennially among the highest in the nation due to a continuous influx of immigrants from the Western Pacific and Asia. TB in Hawaii is composed of a unique distribution of genetic lineages, with the Beijing and Manila families of Mycobacterium tuberculosis (Mtb) comprising over two-thirds of TB cases. Standard fingerprinting methods (spoligotyping plus 24-loci Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats [MIRU-VNTR] fingerprinting) perform poorly when used to identify actual transmission clusters composed of isolates from these two families. Those typing methods typically group isolates from these families into large clusters of non-linked isolates with identical fingerprints. Next-generation whole-genome sequencing (WGS) provides a new tool for molecular epidemiology that can resolve clusters of isolates with identical spoligotyping and MIRU-VNTR fingerprints. METHODS: We performed WGS and SNP analysis and evaluated epidemiological data to investigate 19 apparent TB transmission clusters in Hawaii from 2003 to 2017 in order to assess WGS' ability to resolve putative Mtb clusters from the Beijing and Manila families. This project additionally investigated MIRU-VNTR allele prevalence to determine why standard Mtb fingerprinting fails to usefully distinguish actual transmission clusters from these two Mtb families. RESULTS: WGS excluded transmission events in seven of these putative clusters, confirmed transmission in eight, and identified both transmission-linked and non-linked isolates in four. For epidemiologically identified clusters, while the sensitivity of MIRU-VNTR fingerprinting for identifying actual transmission clusters was found to be 100%, its specificity was only 28.6% relative to WGS. We identified that the Beijing and Manila families' significantly lower Shannon evenness of MIRU-VNTR allele distributions than lineage 4 was the cause of standard fingerprinting's poor performance when identifying transmission in Beijing and Manila family clusters. CONCLUSIONS: This study demonstrated that WGS is necessary for epidemiological investigation of TB in Hawaii and the Pacific.

Geographical differences associated with single-nucleotide polymorphisms (SNPs) in nine gene targets among resistant clinical isolates of Mycobacterium tuberculosis.

Alternative diagnostic methods, such as sequence-based techniques, are necessary for increasing the proportion of tuberculosis cases tested for drug resistance. Despite the abundance of data on drug resistance, isolates can display phenotypic resistance but lack any distinguishable markers. Furthermore, because resistance-conferring mutations develop under antibiotic pressure, different drug regimens could favor unique single-nucleotide polymorphisms (SNPs) in different geographical regions. A total of 407 isolates were collected from four geographical regions with a high prevalence of drug-resistant tuberculosis (India, Moldova, the Philippines, and South Africa). The "hot spot" or promoter sequences of nine genes (rpoB, gyrA, gyrB, katG, inhA promoter, ahpC promoter, eis promoter, rrs, and tlyA) associated with resistance to four types of antibiotics (rifampin, isoniazid, fluoroquinolones, and aminoglycosides) were analyzed for markers. Four genes contributed largely to resistance (rpoB, gyrA, rrs, and katG), two genes contributed moderately to resistance (the eis and inhA promoters), and three genes contributed little or no resistance (gyrB, tlyA, and the ahpC promoter) in clinical isolates. Several geographical differences were found, including a double mutation in rpoB found in 37.1% of isolates from South Africa, the C→T mutation at position -12 of the eis promoter found exclusively in 60.6% of isolates from Moldova, and the G→A mutation at position -46 of the ahpC promoter found only in India. These differences in polymorphism frequencies emphasize the uniqueness of isolates found in different geographical regions. The inclusion of several genes provided a moderate increase in sensitivity, and elimination of the examination of other genes might increase efficiency.

Predicting extensively drug-resistant Mycobacterium tuberculosis phenotypes with genetic mutations.

Molecular diagnostic methods based on the detection of mutations conferring drug resistance are promising technologies for rapidly detecting multidrug-/extensively drug-resistant tuberculosis (M/XDR TB), but large studies of mutations as markers of resistance are rare. The Global Consortium for Drug-Resistant TB Diagnostics analyzed 417 Mycobacterium tuberculosis isolates from multinational sites with a high prevalence of drug resistance to determine the sensitivities and specificities of mutations associated with M/XDR TB to inform the development of rapid diagnostic methods. We collected M/XDR TB isolates from regions of high TB burden in India, Moldova, the Philippines, and South Africa. The isolates underwent standardized phenotypic drug susceptibility testing (DST) to isoniazid (INH), rifampin (RIF), moxifloxacin (MOX), ofloxacin (OFX), amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) using MGIT 960 and WHO-recommended critical concentrations. Eight genes (katG, inhA, rpoB, gyrA, gyrB, rrs, eis, and tlyA) were sequenced using Sanger sequencing. Three hundred seventy isolates were INHr, 356 were RIFr, 292 were MOXr/OFXr, 230 were AMKr, 219 were CAPr, and 286 were KANr. Four single nucleotide polymorphisms (SNPs) in katG/inhA had a combined sensitivity of 96% and specificities of 97 to 100% for the detection of INHr. Eleven SNPs in rpoB had a combined sensitivity of 98% for RIFr. Eight SNPs in gyrA codons 88 to 94 had sensitivities of 90% for MOXr/OFXr. The rrs 1401/1484 SNPs had 89 to 90% sensitivity for detecting AMKr/CAPr but 71% sensitivity for KANr. Adding eis promoter SNPs increased the sensitivity to 93% for detecting AMKr and to 91% for detecting KANr. Approximately 30 SNPs in six genes predicted clinically relevant XDR-TB phenotypes with 90 to 98% sensitivity and almost 100% specificity.

Rapid deletion-based subtyping system for the Manila family of Mycobacterium tuberculosis.

The Manila family of Mycobacterium tuberculosis is a group of clonal isolates seen throughout the Pacific Basin. Commonly used rapid molecular typing methods often leave large groups of Manila family isolates clustered together. Here we describe a simple deletion-based PCR method that improves the discrimination for Manila family isolates, with or without the use of mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) classification, and that is both rapid and affordable. Twenty-eight Manila family isolates, classified by spoligotyping, were collected from around the Pacific Basin from 1995 to 2003 and were tested for known genomic deletions. Nine of 15 regions of difference tested were identified as potentially discriminatory, with 18 distinct patterns; of these 9, 5 were selected for optimal discrimination using 61 Manila family isolates collected from California in 2009. For this geographically limited sample, the single large cluster was reduced to 14 distinct patterns. When the isolates were tested by spoligotyping and MIRU-VNTR, the addition of deletion analysis increased the number of distinct patterns from 43 to 56. In summary, the two study groups, which together form a single group of 89 isolates by spoligotyping, were segregated into 17 subgroups by our deletion-based subtyping system.

Whole genome SNP analysis suggests unique virulence factor differences of the Beijing and Manila families of Mycobacterium tuberculosis found in Hawaii.

While tuberculosis (TB) remains a global disease, the WHO estimates that 62% of the incident TB cases in 2016 occurred in the WHO South-East Asia and Western Pacific regions. TB in the Pacific is composed predominantly of two genetic families of Mycobacterium tuberculosis (Mtb): Beijing and Manila. The Manila family is historically under-studied relative to the families that comprise the majority of TB in Europe and North America (e.g. lineage 4), and it remains unclear why this lineage has persisted in Filipino populations despite the predominance of more globally successful Mtb lineages in most of the world. The Beijing family is of particular interest as it is increasingly associated with drug resistance throughout the world. Both of these lineages are important to the State of Hawaii, where they comprise over two-thirds of TB cases. Here, we performed whole genome sequencing on 82 Beijing family, Manila family, and outgroup clinical Mtb isolates from Hawaii to identify lineage-specific SNPs (SNPs found in all isolates from their respective families, and exclusively in those families) in established virulence factor genes. Six non-silent lineage-specific virulence factor SNPs were found in the Beijing family, including mutations in alternative sigma factor sigG and polyketide synthases pks5 and pks7. The Manila family displayed more than eleven non-silent lineage-specific and characteristic virulence factor mutations, including in genes coding for MCE-family protein Mce1B, two mutations in fatty-acid-AMP ligase FadD26, and virulence-regulating transcriptional regulator VirS. This study further identified an ancient clade that shared some virulence factor mutations with the Manila family, and investigated the relationship of those and other "Manila-like" spoligotypes to the Manila family with this SNP dataset. This work identified a set of virulence genes that are worth pursuing to determine potential differences in transmission or virulence displayed by these two Mtb families.

Genomic analyses of the ancestral Manila family of Mycobacterium tuberculosis.

With its airborne transmission and prolonged latency period, Mycobacterium tuberculosis spreads worldwide as one of the most successful bacterial pathogens and continues to kill millions of people every year. M. tuberculosis lineage 1 is inferred to originate ancestrally based on the presence of the 52-bp TbD1 sequence and analysis of single nucleotide polymorphisms. Previously, we briefly reported the complete genome sequencing of M. tuberculosis strains 96121 and 96075, which belong to the ancient Manila family and modern Beijing family respectively. Here we present the comprehensive genomic analyses of the Manila family in lineage 1 compared to complete genomes in lineages 2-4. Principal component analysis of the presence and absence of CRISPR spacers suggests that Manila isolate 96121 is distinctly distant from lineages 2-4. We further identify a truncated whiB5 gene and a putative operon consisting of genes encoding a putative serine/threonine kinase PknH and a putative ABC transporter, which are only found in the genomes of Manila family isolates. Six single nucleotide polymorphisms are uniquely conserved in 38 Manila strains. Moreover, when compared to M. tuberculosis H37Rv, 59 proteins are under positive selection in Manila family isolate 96121 but not in Beijing family isolate 96075. The unique features further serve as biomarkers for Manila strains and may shed light on the limited transmission of this ancestral lineage outside of its Filipino host population.

Complete Genome Sequences of Beijing and Manila Family Strains of Mycobacterium tuberculosis.

The majority of isolates from tuberculosis patients in Hawaii arrive through the immigration of infected individuals from the western Pacific. We report here on the annotated complete genomes of two strains of Mycobacterium tuberculosis from the two main lineages/families in Hawaii, Beijing and Manila.

rpoB genotypes of Mycobacterium tuberculosis Beijing family isolates from East Asian countries.

The 81-bp region of the rpoB gene in 66 Rif(r) Mycobacterium tuberculosis isolates from China, Japan, Korea, and Taiwan was analyzed. Twelve single-nucleotide substitutions in the rpoB gene were detected. The most prevalent mutations were at Ser-531 (52%), Asp-516 (17%), and His-526 (11%). Mutations were not found in seven (11%) of the isolates. Higher mutation rates in 50 Beijing family isolates were found than in other isolates for mutations at Asp-516 (18 and 12.5%, respectively) and His-526 (12 and 6.3%, respectively). The different rates of mutation may reflect the choice of rifamycin analogs.

Characterization of the Manila family of Mycobacterium tuberculosis.

Forty-eight Mycobacterium tuberculosis strains were obtained from patients living in metropolitan Manila, Republic of the Philippines. Three molecular typing methods, IS6110 restriction fragment length polymorphism, spoligotyping, and DNA sequencing of the oxyR, gyrA, and katG loci, established that these strains have restricted diversity and are members of a related genetic group of organisms. Comparison of the DNA fingerprint patterns with those in international databases confirmed the uniqueness of this group of isolates, which we designate the Manila family of M. tuberculosis.